The Effects of Gatorade and Salinity on Biofilms

THE EFFECTS OF GATORADE AND SALINITY ON BIOFILMS Renne Cabacungan Grade 11 Central Catholic High School GATORADE Used for rapid replacement of electrolytes and fluid lost during exercise. Restores depleted muscle carbohydrate storages that cause muscle failure and fatigue. GATORADE INGREDIENTS Water Glucose Sucrose Dextrose Total Carbohydrates 34g Citric acid Natural flavor Sodium citrate Monopotassium phosphate

Gum arabic Red 40 Glycerol ester of rosin Protein 0g Sodium 270 mg Potassium 75mg Total Fat 0g MORTONS TABLE SALT Serving size: cup; 560 mg Sodium Solar Evaporation Extraction & Mine Production Iodized salt Salinity and cellular hypertonicity

BIOFILMS Coherent and generally adherent cells Extracellular Polymeric Substance/Extracellular Matrix Phenotypic shift in gene regulation Lateral gene transfer BIOFILM INHIBITION Adherence- conditioning films, polysaccharides Anti-biofilm agents: chemical inhibitors to adherence and activity o Anti-microbial surface conditioning Hydrophobic Nontoxic biofilm agents STAPHYLOCOCCUS EPIDERMIDIS Bacteria usually non-pathogenic towards humans Commonly found on the epidermis of humans Common prokaryote model Gram + bacteria RATIONALE/APPLICATION Biofilms account for 80% of infectious disease Developing information on biofilm formation/inhibition

Past studies of Gatorade/Salinity antimicrobial effects Medical implications: o Biofilm targeted disinfectants o Potential for salinity treatments PURPOSE To determine the effects of Gatorade on biofilm formation To determine the effects of salt on biofilm formation To test for an interactive effect amongst the two variables To test for relationship with antimicrobial effects HYPOTHESIS Null Hypothesis: Salt and Gatorade will not have a significant effect on Staph epidermidis biofilm formation and survivorship. Alternative Hypothesis: Salt and Gatorade alone will have a significant effect on Staph epidermidis biofilm formation and survivorship. Salt and Gatorade together will have an interactive effect on biofilm formation and survivorship. MATERIALS Staph. E culture (Wards Science) Gatorade

Table Salt LB agar plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile dilution fluid (100 mM KH2PO4, 100 mM K2HPO4, 10 mM MgSO4, 1mM NaCl) Sterile pipette tips Micropipettes Vortex Incubator (37 C) Sidearm Flask Sterile Spreader Bars Ethanol 96 well tissue culture treated microtiter dish Crystal Violet Acetic Acid Microtiter plate absorbance reader SURVIVORSHIP PROCEDURE 1. Staph. E was grown overnight in sterile LB Media.

2. The culture was added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 cells/mL. 5. A solution of salt and SDF were created as a 20% stock. 6. The Gatorade and salt water were sterilized by means of a 0.2 micron syringe filter 7. Experimental variables were mixed with the appropriate amounts of SDF to create concentrations. CONCENTRATION CHART Gatorade Salt Solution SDF Bacteria (Staph) Concentratio n

0 mL 0 mL 9.9 mL 0.1 mL 0% G 0% S 0.5 mL 0 mL 9.4 mL 0.1 mL 5% G 0% S 2 mL

0 mL 7.9 mL 0.1 mL 20% G 0% S 0 mL 0.1 mL 9.8 mL 0.1 mL 0% G 1% S 0 mL 0.5 mL 9.4 mL

0.1 mL 0% G 5% S 0.5 mL 0.1 mL 9.3 mL 0.1 mL 5% G 1% S 0.5 mL 0.5 mL 8.9 mL 0.1 mL

5 % G 5% S 2 mL 0.1 mL 7.8 mL 0.1 mL 20% G 1% S 2 mL 0.5 mL 7.4 mL 0.1 mL 20% G 5% S CONCENTRATIONS SIMPLIFIED

Gatorade Salt 0 Low (5%) High (20%) 0 00 0L 0H Low (1%) L0 LL

LH High (5%) H0 HL HH PROCEDURE (CONT.) 8. The solutions were vortexed and allowed to sit at room temperature for 10 minutes. 9. 100 L aliquots were removed from the tubes and spread on LB-agar plates. 10. The plates were incubated at 37C for 48 hours. 11. The resulting colonies were counted visually (each colony was assumed to have arisen from one cell) 12. Steps were repeated for 7 replicates. BIOFILM FORMATION Growing a Biofilm 1. Tubes were prepared according to the concentration chart (scaled down to

microtube volume of 2mL) 2. 200 L from the tubes was added per well in a 96 well dish. 8 replicates were performed from each tube. 3. The microtiter plate was incubated for 48 hours at 37C. Staining the Biofilm 1. After incubation, the cells were gently removed out by turning the plate and allowing to drip dry. 2. The plate was gently submerged in a small tub of water. The plate was allowed to drip dry. 3. 200 L of a 0.1% solution of crystal violet in water was added to each well of the BIOFILM FORMATION (CONT.) 4. The microtiter plate was incubated at room temperature for 10 minutes. 5. The plate was rinsed by submerging in a tub of water as outlined above. 6. The microtiter plate was turned upside down and dried overnight. Quantifying the Biofilm 1. 200 L of 30% acetic acid in water was added to each well of the microtiter plate to solubilize the CV. 2. The microtiter plate was incubated at room temperature for 10 minutes. plate reader at 550 nm using 30% acetic acid in water as the blank. 3. The absorbance of the microtiter plates was quantified in a microtiter 450

400 Gatorade & Salt Concentrations vs. Survivorship 387 P Value: 0.0192 373 Resulting colonies 350 323 332 298 300 269 254 250

0% Salt 5% Salt 260 200 144 150 100 50 0 0% Gatorade 5% Gatorade 20% Gatorade Variable Concentrations 1% Salt

Absorbance (L/ Molcm) Gatorade & Salinity Vs. Biofilm Formation P Value: 0.02145 4 3.63 3.61 3.5 3.55 3.5 3.51 3.44 3.25

3.11 0% Salt 3 2.42 2.5 2 1.5 1 0.5 0 0% Gatorade 5% Gatorade 20% Gatorade Variable Concentrations

1% Salt Percent Reduction 70 63 60 % Reduction 50 40 34 30 30 0% G 1% S 0% G 5% S

5% G 0% S 5% G 1% S 5% G 5% S 20% G 0% S 20% G 1% S 20% G 5% S 34 33 23 20 10 0

16 14 4 Survivorship 12 3 4 4 0.68 6 6 Biofilm Experimental procedure CONCLUSIONS Survivorship: The data supports the rejection of the null hypothesis, only in the presence of high salt and Gatorade content, possibly implying a synergistic effect. Biofilm: The data supports the rejection of the null hypothesis, only in the presence of high salt and Gatorade content, possibly implying a synergistic effect. In lower concentrations, the null hypothesis is accepted, the data does not

support a significant effect in both cases, for lower concentrations. LIMITATIONS & EXTENSIONS Limitations: Extensions: Only one exposure time More Replicates Technique Errors Multiple Model organisms Only Inhibition/Survivorship tested Test Reproduction/Promotion Limited Replicates More Concentrations

Only Tested One Model Organism Different Types of Salt/Different Sports Drinks Reliability of biofilm assay SOURCES https://www.future-science.com/doi/full/10.4155/fmc.15.7 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146668 https://www.livestrong.com/article/43161-list-ingredients-gatorade/ http://www.mortonsalt.com/article/morton-iodized-table-salt-nutritional-facts/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732559/ https://www.ncbi.nlm.nih.gov/pubmed/23635385 *Dr. Carrie Doonan, CMU, for lab space and equiptment SURVIVORSHIP ANOVA ANOVA Source of VariationSS Sample 161232.8 Columns 127873.6 Interaction 12791.11

Within 53619.14 Total 355516.6 df 2 2 4 54 62 MS F P-value F crit 80616.4 81.18902 5.3E-17 3.168246 63936.78 64.39092 5.04E-15 3.168246 3197.778 3.220492 0.019218 2.542918 992.9471 BIOFILM ANOVA

ANOVA Source of VariationSS Sample 3.207414 Columns 3.038611 Interaction 2.71227 Within 14.14244 Total 23.10073 df 2 2 4 63 71 MS F P-value F crit

1.603707 7.143998 0.001598 3.142809 1.519306 6.768016 0.002175 3.142809 0.678068 3.020572 0.02415 2.51767 0.224483

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